Analytix_3_06_rz.indd

Mobile Phase Additives for LC-MS. Part 2: How to Overcome Suppression Effects of
TFA . This is the second installment in a five-part series on mobile phase additives for LC-MS to
appear in each of the issues of Analytix in 2006.
By Joachim Emmert, Senior Scientist R&D Fluka / Riedel-de Haën … [email protected]
and Alex Rueck, Scientist R&D Fluka / Riedel-de Haën … [email protected]
Mobile phases for HPLC of proteins and peptides usually TFA has adverse effects on MS detection. Its high contain trifluoroacetic acid (TFA) to control the pH and surface tension prevents efficient spray formation and improve peak shape and resolution. TFA enhances TFA ions in the gas phase ion-pair with the peptide’s retention by ion pairing with the peptide and improves basic groups suppressing their ionization and reducing peak shape by reducing silanol interactions (1). However, the MS signal (2, 3, 4). When TFA cannot be avoided, its effects can be mitigated by additional use of other acids, Table 1 . LC-MS additives
like formic or propionic acid, either post-column or as so Cat. No. Brand Description*
Package Size Packaging
called triple blends (Tables 1 and 2).
Trifluoroacetic acid, puriss p.a., eluent 50 mL All analytical conditions and test compounds were the Trifluoroacetic acid, puriss p.a., eluent 10 x 1 mL same as already described in the first article (5), using TFA as additive instead or the triple blends as solvents Formic acid, puriss p.a., eluent additive respectively. Propionic acid was added post column / pre electrospray via T-piece as a 10% solution in 2-propanol. Acetic acid, puriss p.a., eluent additive For additional experiments, a peptide mixture (pepmix) was prepared to study the specific influence on this kind of separation. The test compounds and the pepmix were both separated on a Supelco Discovery HS C18 column, 15 cm x 2.1 mm ID, 5 µm particle size; the 5 components (peptides) of the pepmix are listed in Table 3. MS-EIC
traces are the same for all chromatograms.
Sodium citrate tribasic dihydrate, puriss Figure 1 shows the separation without any additive.
Under these conditions, the basic peptide bradykinin is barely distinguishable from the baseline. Its mass spectrum can still be obtained (Figure 2, lower),
showing the doubly-charged molecular ion [M+2H]2+ Triethylamine, puriss p.a., eluent additive 50 mL with m=1061.6 or m/z = 530.8. Raffinose is unaffected by adding TFA or other organic acids. Its spectrum *”puriss” quality grade is defined as >98.5% assay, <0.1% ash, and specification n + 0.001, d + 0.001 with no extraneous color and a homogeneous appearance. “p.a.” or pro analysi denotes (Figure 2, upper) shows the H+ (505 m/z) and NH +
a product with guaranteed trace impurity levels and/or suitability for the indicated analytical adducts (522.1 m/z) and the high abundance Na+ adduct Table 2 . Selection of LC-MS solvents and blends
Cat. No. Brand
Solvent or Blend Description
Package Size Packaging
Addition of 0.1% TFA (Figure 3, top) causes all five test
compounds to elute as well separated and sharp peaks. However, note that sensitivity drops almost 10-fold. The suppression effect is reduced by using 0.1% TFA and adding propionic acid (10% in 2-propanol) post-column (Figure 3, middle), an effect described in detail by Apffel
et al. (2). Using the triple-blend of 0.1% formic Table 3 . Components of the peptide mixture
acid/0.01% TFA (Figure 3, lower) greatly improves the
Component
Molecular Mass
Mol. ion / Charge
signal, but with a compromise. Compared to TFA alone, resolution is poorer; and compared to formic acid alone The three additives can be used in synergy, by balancing their benefits and limitations. Add small amounts of TFA to formic or propionic acid to improve peak shape; www.sigma-aldrich.com/lc-ms-additives
Figure 1 .
reduce TFA and add formic or propionic acid to improve the MS signal. Other MS and chromatographic para- meters also influence this decision, including analytes, column packing material and dimensions, length of mixing zone, flow rate, etc. (1, 2). This is especially true for peptide separations. The charge state of the molecular ion is not affected by this and varies in the bradykinin (green) is not eluting as a peak pepmix between singly-charged (Bradykinin fragment 1-7) and triply-charged (insulin oxidized B chain)
(Table 3). Depending on instrument and conditions it
may be the case that one peptide appears in more than one charge state, i.e. doubly- and triply-charged in one Figure 2 .
spectrum (Figure 4).
In summary, the ionization-suppressing effects of TFA can be partly overcome by addition of other LC-MS bradykinin (lower) without additive.
compatible organic acids, like formic or propionic acid. For convenience and to guarantee reliable composition, Sigma-Aldrich offers pre-blended LC-MS mobile phases that contain acidic additives in high purity LC-MS CHROMASOLV® grade solvents. Our triple blends contain TFA with formic acid to provide both MS sensitivity and chromatographic performance. Figure 3 .
Figure 4 .
Mass Spectrum of ACTH fragm. 18-39 with 0.1% TFA as additive and 10% propionic acid post-column. Doubly (m/z 1233.1) and triply (m/z 822.4) charged molecular ions.
propionic acid added post-column (middle) (lower) as additive. EIC traces are the same References
[1] “Eliminate TFA and Improve Sensitivity of Peptide Analyses by
LC-MS” Supelco Application Note 168 (T302168).
[2] A. Apffel, A.; Fisher, S.; Goldberg, G.; Goodley, P. C.; Kuhlmann, F.E.; J. Chromatogr. A, 1995, 712, 177-190.
[3] Mirza, U. A.; Chait, B. T.; Anal. Chem. 1994, 66, 2898-2904.
[4] Wang, G; Cole, R. B.; J. Am. Soc. Mass Spectrom., 1996, 7(10), [5] “Mobile Phase Additives for LC-MS. Part 1: Acids – The Most Common Choice” Analytix 2006/2, 8-9.
www.sigma-aldrich.com/lc-ms-additives

Source: http://www.emmert-analytik.de/Add3_LC-MS.pdf